A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Comodulation of CXCR4 and CD26 in human lymphocytes

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.     

Cloning and expression of the CD4 receptor gene from human T-lymphocytes in Escherichia coli cells

The gene for the CD4-membrane glycoprotein-receptor for HIV has been cloned. The 179 amino acids fragment of the CD4-receptor responsible for binding of gp120 HIV glycoprotein has been fused with beta-galactosidase and shown to be expressed in Escherichia coli cells. The recombinant protein in ELISA and immunoblotting techniques reacts with the monoclonal antibodies OKT4A and Leu3A known to block the interaction between the CD4 and gp120 HIV glycoprotein. The recombinant protein can be used for different scientific and practical purposes including studying of the mechanisms for HIV interaction with the sensitive cells as well as for viral gp120 protein purification, etc.     

Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.     

Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding.

Conflicting results have been reported regarding the role of carbohydrate on human immunodeficiency virus (HIV) envelope glycoprotein gp120 in CD4 receptor binding. Glycosylated, deglycosylated, and nonglycosylated forms of HIV type 1 (HIV-1) and HIV-2 gp120s were used to examine CD4 receptor-binding activity. Nonglycosylated forms of gp120 generated either by deletion of the signal sequence of HIV-1 gp120 or by synthesis in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated gp120 bound to soluble CD4 molecules well. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H or an endoglycosidase F/N glycanase mixture had no effect on the ability of gp120 to bind CD4. An experiment which measured the ability of gp120 to bind to CD4 as an assay of the proper conformation of gp120 showed that carbohydrate chains on gp120 are not required for the interaction between gp120 and CD4 but that N-linked glycosylation is essential for generation of the proper conformation of gp120 to provide a CD4-binding site.     

CD4 epitope masking by gp120/anti-gp120 antibody complexes. A potential mechanism for CD4+ cell function down-regulation in AIDS patients.

The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency.     

Evidence by peptide mapping that the region CD4(81-92) is involved in gp120/CD4 interaction leading to HIV infection and HIV-induced syncytium formation.

Peptide fragments of the CD4 molecule were compared in their ability to 1) inhibit CD4-dependent HIV-induced cell fusion; 2) inhibit CD4-dependent HIV infection in vitro; and 3) block gp120 envelope glycoprotein binding to CD4. Peptides from the region CD4(81-92), although inactive when underivatized, were equipotent inhibitors of CD4-dependent virus infection, cell fusion, and CD4/gp120 binding when derivatized via benzylation and acetylation. Peptides of identical chemical composition, but altered sequence and derivatization pattern that blocked gp120 binding to either CD4-positive cells or solubilized CD4, also blocked infection and fusion with similar potencies. Those that did not block gp120/CD4 interaction were also inactive in HIV-1 infection and cell fusion assays. No other peptide fragments of the CD4 molecule inhibited fusion, infection, or CD4/gp120 interaction. The peptide CD4(23-56), derived from a region of CD4 implicated in binding of CD4 antibodies that neutralize HIV infection and cell fusion, had no effect on CD4-dependent cell fusion, HIV-1 infection, or CD4/gp120 binding, but did reverse OKT4A and anti-Leu 3a blockade of gp120 binding to CD4. These data provide evidence that the 81-92 region of CD4 is directly involved in gp120 binding leading to CD4-dependent HIV infection and syncytium formation. Previous observations with structural mutants of CD4 suggest that the CDR2-homologous region of CD4 is also involved, either directly or indirectly, in binding of gp120 to CD4. The CDR2- and CDR3-like domains of CD4 may both contribute to the binding of the HIV envelope necessary for HIV-1 infection and HIV-1-induced cell fusion.     

Analysis of the site in CD4 that binds to the HIV envelope glycoprotein.

The first step in infection of human mononuclear cells with HIV involves the high affinity binding of the viral envelope glycoprotein, gp120, to the cell-surface receptor, CD4. To gain a better understanding of the molecular basis of this interaction, we have analyzed the ability of gp120 to bind to a panel of 40 mutant CD4 proteins containing single or double amino acid substitutions. In addition, the binding of several anti-CD4 mAb to the mutant CD4 proteins was measured. These mAb were chosen on the basis of the previous demonstration that they bind to epitopes in CD4 adjacent to the gp120-binding site. This analysis permits discrimination between mutations that probably cause localized conformational changes and those that alter residues likely to make direct contact with gp120 and with the mAb. Our results indicate that gp120 from two different strains of HIV binds to a larger region of the CD4 protein than previously described. The data has also been used to map the epitopes of mAb previously identified as anti-idiotype vaccine candidates. The results have important implications for the development of CD4-based therapies for AIDS.     

Trypsin-resistant gp120 receptors are upregulated on short-term cultured human epidermal Langerhans cells

The CD4 molecule is known to be the preferential receptor for the HIV1 envelope glycoprotein. Epidermal Langerhans cells (LC) are dendritic cells which express several surface antigens, among them the CD4 antigens. LC infection was suggested when these cells were seen to present buddings coincident with membrane thickening of roughly 100 nm in size. These buddings were similar in ultrastructural aspect to HIV buddings on in vitro infected promonocytic cells (U937). To clarify the exact role of CD4 molecules in LC infection induced by HIV1, we investigated the possible involvement of the interactions between native and recombinant HIV1 gp 120 and the LC surface. We also assessed the expression of CD4 molecules on LC membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry. We show that human LC can bind the viral envelope protein and that this binding does not depend on CD4 protein expression. The amount of surface bound gp120 was not consistent with the amount of CD4 antigens present on LC membranes. The gp 120-binding sites on LC in suspension appear to be trypsin-resistant while the CD4 antigens (at least the epitopes known to bind HIV1) are trypsin-sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured LC while the CD4 antigens disappeared. These findings lead to the logical conclusion that the binding of gp 120 is due to the presence of a LC surface molecule which is different from CD4 antigens.     

Structural mimicry of CD4 by a cross-reactive HIV-1 neutralizing antibody with CDR-H2 and H3 containing unique motifs

Human immunodeficiency virus (HIV) entry into cells is initiated by the binding of its envelope glycoprotein (Env) gp120 to receptor CD4. Antibodies that bind to epitopes overlapping the CD4-binding site (CD4bs) on gp120 can prevent HIV entry by competing with cell-associated CD4; their ability to outcompete CD4 is a major determinant of their neutralizing potency and is proportional to their avidity. The breadth of neutralization and the likelihood of the emergence of antibody-resistant virus are critically dependent on the structure of their epitopes. Because CD4bs is highly conserved, it is reasonable to hypothesize that antibodies closely mimicking CD4 could exhibit relatively broad cross-reactivity and a high probability of preventing the emergence of resistant viruses. Previously, in a search for antibodies that mimic CD4 or the co-receptor, we identified and characterized a broadly cross-reactive HIV-neutralizing CD4bs human monoclonal antibody (hmAb), m18. Here, we describe the crystal structure of Fab m18 at 2.03 A resolution, which reveals unique conformations of heavy chain complementarity-determining regions (CDRs) 2 and 3 (H2 and H3). H2 is highly bulged and lacks cross-linking interstrand hydrogen bonds observed in all four canonical structures. H3 is 17.5 A long and rigid, forming an extended beta-sheet decorated with an alpha-turn motif bearing a phenylalanine-isoleucine fork at the apex. It shows striking similarity to the Ig CDR2-like C'C' region of the CD4 first domain D1 that dominates the binding of CD4 to gp120. Docking simulations suggest significant similarity between the m18 epitope and the CD4bs on gp120. Fab m18 does not enhance binding of CD4-induced (CD4i) antibodies, nor does it induce CD4-independent fusion mediated by the HIV Env. Thus, vaccine immunogens based on the m18 epitope structure are unlikely to elicit antibodies that could enhance infection. The structure can also serve as a basis for the design of novel, highly efficient inhibitors of HIV entry.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.     

Homology models of the HIV‐1 attachment inhibitor BMS‐626529 bound to gp120 suggest a unique mechanism of action

HIV‐1 gp120 undergoes multiple conformational changes both before and after binding to the host CD4 receptor. BMS‐626529 is an attachment inhibitor (AI) in clinical development (administered as prodrug BMS‐663068) that binds to HIV‐1 gp120. To investigate the mechanism of action of this new class of antiretroviral compounds, we constructed homology models of unliganded HIV‐1 gp120 (UNLIG), a pre‐CD4 binding‐intermediate conformation (pCD4), a CD4 bound‐intermediate conformation (bCD4), and a CD4/co‐receptor‐bound gp120 (LIG) from a series of partial structures. We also describe a simple pathway illustrating the transition between these four states. Guided by the positions of BMS‐626529 resistance substitutions and structure–activity relationship data for the AI series, putative binding sites for BMS‐626529 were identified, supported by biochemical and biophysical data. BMS‐626529 was docked into the UNLIG model and molecular dynamics simulations were used to demonstrate the thermodynamic stability of the different gp120 UNLIG/BMS‐626529 models. We propose that BMS‐626529 binds to the UNLIG conformation of gp120 within the structurally conserved outer domain, under the antiparallel β20–β21 sheet, and adjacent to the CD4 binding loop. Through this binding mode, BMS‐626529 can inhibit both CD4‐induced and CD4‐independent formation of the “open state” four‐stranded gp120 bridging sheet, and the subsequent formation and exposure of the chemokine co‐receptor binding site. This unique mechanism of action prevents the initial interaction of HIV‐1 with the host CD4+ T cell, and subsequent HIV‐1 binding and entry. Our findings clarify the novel mechanism of BMS‐626529, supporting its ongoing clinical development. Proteins 2015; 83:331–350. © 2014 Wiley Periodicals, Inc.     

Binding of full-length HIV-1 gp120 to CD4 induces structural reorientation around the gp120 core

Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor, and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.     

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.     

HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases

Background

During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR) as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs) downstream of HIV gp120 binding to hMR.

Principal Findings

Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line) and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody.

Conclusion

hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the vaginal epithelium.     

Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis

The initial step of infection of blood cells with the human immunodeficiency virus, HIV, is the formation of a complex of the viral envelope protein gp120 and its human receptor CD4. We have examined structural features of recombinant soluble CD4 (sCD4) by chemical modification of arginine residues with hydroxyphenylglyoxal and subsequent analysis by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. As R58, R59, R131, R134, R219, R240, R293, and R329 could be derivatized free in solution, these arginine residues were exposed on the surface of the protein. In the noncovalent complex of sCD4 with HIV(SF2)gp120, only R58, R131, R134, R219, R240, R293, and R329 were accessible for the derivatizing agent. R59 was shielded from hydroxyphenylglyoxal and was, therefore, considered to be part of the interaction site with gp120. This indicates that the carbohydrate moieties and the flexible variable loops of the glycosylated full-length gp120 from HIV strain SF2 do not induce a reorganization of CD4 in its binding to gp120 and, therefore, do not appear to significantly affect the structural orientation of the primary receptor in complex with the HIV envelope protein as compared to the binding observed in the crystal structure of CD4 with truncated deglycosylated gp120.     

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.     

Dissociation of the CD4 and CXCR4 Binding Properties of Human Immunodeficiency Virus Type 1 gp120 by Deletion of the First Putative Alpha-Helical Conserved Structure

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 ΔαHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1α, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4⁺/CD4⁻ cells but not to CXCR4⁻/CD4⁻ cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 ΔαHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4⁺ cells. Nevertheless, the soluble gp120 ΔαHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking αHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 ΔαHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.     

Fluctuation dynamics analysis of gp120 envelope protein reveals a topologically based communication network

Human Immunodeficiency Virus (HIV) infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. On CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of the Gaussian network model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. These results provide a new context for interpreting gp120 core envelope structure–function relationships. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.